This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Site-directed spin labeling (SDSL) is a powerful tool for monitoring the structure and dynamics of both soluble and membrane proteins. Measurements of the spectral properties of the paramagnetic nitroxide probe with electron paramagnetic resonance (EPR) spectroscopy provide a wealth of information on the environment in the protein. From the EPR spectrum of the spin-bearing moiety, four primary parameters are obtained: (i) solvent accessibility ([unreadable]);(ii) mobility (Ms) of the spin-label side chain;(iii) a polarity index for its immediate environment;and (iv) the distance between R1 and another paramagnetic center in the protein (r), which can be either a second nitroxide or a metal ion. Changes in [unreadable], Ms, the polarity index, and r can all be used to detect changes in protein conformations, and, most importantly, the data can be interpreted in terms of helix rigid body motions, relative domain movement, and changes in secondary structure. Moreover, changes in Ms and r can be monitored in real time in the millisecond timescale with conventional EPR instrumentation. Multifrequency SDSL studies are currently underway to investigate the energy-coupling segment of BtuB, a 66 kDa porin-like outer membrane protein found in Gram-negative bacteria such as Escherichia coli. BtuB transports its substrate, vitamin B12, across the outer membrane using free energy obtained by coupling to the inner membrane protein TonB. Preliminary SDSL studies at several sites along the TonB box sequence exhibit lineshapes with multiple components typical of side chains with strong tertiary interaction,whereas other sites yield lineshapes characteristic of exposed side chains. We are initiating a multifrequency study of this important membrane protein to gather further insights into the details of the dynamic modes that inform its function.